Project Summary Transcription is a fundamental process that mediates the interplay between genetic information and phenotypes, thus vital for development, responses to environmental cues, and diseases. Transcription is primarily catalyzed by DNA-directed RNA polymerases (DdRPs), which are highly conserved among eukaryotic organisms. It has been well established that regulation from multiple layers controls DdRP- catalyzed transcription on DNA templates. Interestingly, some DdRPs recognize both DNA and RNA templates for transcription. This RNA-templated activity regulates gene expression in bacteria and mammalian cells and is employed by pathogens (i.e., viroids and human hepatitis delta virus) for propagation. However, the machinery and mechanism for RNA-templated transcription by DdRPs are poorly understood. Using the replication of potato spindle tuber viroid (PSTVd) as a model, preliminary data showed that a highly conserved glycine in the second largest subunit of Pol II is critical for DNA-directed but not RNA-templated transcription, indicating the different requirements of this residue for DNA and RNA templates. In addition, data showed that TFIIS, a general transcription factor essential for DNA-directed transcription, is dispensable for PSTVd RNA-templated transcription, suggesting that a specialized group of factors are required for transcription of DNA versus RNA templates. Furthermore, the eukaryotic transcription factor TFIIIA-7ZF, which binds RNA but not DNA, has been shown as a critical factor in facilitating Pol II-catalyzed transcription on PSTVd RNA templates. The TFIIIA-7ZF binding site maps to a region next to the transcription initiation site and is critical for Pol II binding and PSTVd replication, suggesting that this binding site acts as an RNA promoter. Together, these findings inspire the central hypothesis that Pol II recruits a specialized group of factors to selectively recognize RNA as templates and catalyze transcription. Using a novel in vitro transcription (IVT) assay that is based on Pol II-catalyzed transcription of PSTVd, the goal of this project is to further dissect the regulatory mechanism for specialized Pol II machinery to transcribe RNA templates. To achieve this goal, three aims are proposed to further dissect the role of TFIIIA-7ZF in RNA-templated transcription, map the TFIIIA-7ZF/Pol II binding domains, and characterize the molecular basis of an RNA promoter. The expected outcomes will provide novel insights into the regulatory mechanism underlying RNA- templated transcription catalyzed by Pol II and provide valuable knowledge to better delineate RNA promoters. As this project aims to uncover the basic principles governing the modus operandi of RNA polymerases that are highly conserved across eukaryotic organisms, the results gained here will provide an in-depth knowledge of RNA-templated transcription. Such in-depth knowledge will lead to the full understanding of factors and the regulatory mechanism underlying RNA-templated transcription and facilitate the development of effective treatments for diseases caused by infectious RNAs.